1. Chapter 17 Synapse Formation Copyright © 2014 Elsevier Inc. All rights reserved.

2. FIGURE 17.1 Pre- and postsynaptic membranes at the neuromuscular synapse are highly specialized. (A) An electron micrograph of a neuromuscular synapse shows that the nerve terminal is capped by a Schwann cell and is situated in a shallow depression of the muscle cell membrane, which is invaginated further into deep and regular folds, termed postjunctional folds (arrows). AChRs, labeled with α-bungarotoxin coupled to horseradish peroxidase, are concentrated at the synaptic site. (B) A higher magnification view shows that AChRs are concentrated at the crests and along the sides of the postjunctional folds (white arrow). Rapsyn, Neuregulin receptors, and MuSK are also concentrated in the postsynaptic membrane, whereas Agrin, Neuregulin-1, acetylcholinesterase, S-laminin, and certain isoforms of collagen are localized to the synaptic basal lamina. The postjunctional folds of the myofiber are spaced at regular intervals and are situated directly across from active zones and clusters of synaptic vesicles in the nerve terminal (arrows).

3. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.2 Muscle prepatterning. (A) Motor axons (green) approach muscles that are prepatterned, as AChR transcription (blue) and AChR clustering (red) are enhanced in the central, prospective synaptic region of muscle, prior to and independent of innervation. Motor axons terminate and form synapses (orange) in the prepatterned region. (B) Lrp4 and MuSK are required for prepatterning AChRs and are themselves expressed in the central region of muscle independent of innervation. The prepatterned zone of AChR expression becomes refined and sharpened following innervation, so that AChR expression and AChR clustering are restricted to nascent synaptic sites. Two neural signals refine AChR expression: Agrin/MuSK signaling stabilizes and stimulates AChR clustering, whereas acetylcholine (ACh) disperses AChR clusters. As a consequence of these two signals, AChR clusters are maintained only at nascent synaptic sites.

4. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.3 The synaptic basal lamina contains signals for presynaptic and postsynaptic differentiation. A cross section of normal muscle containing a nerve terminal, Schwann cell, and myofiber is shown at the far left. AChR genes are induced in synaptic nuclei (red) and AChRs (green) are concentrated at synaptic sites. Active zones and clusters of synaptic vesicles in the nerve terminal are aligned with postjunctional folds in the myofiber. Each myofiber is ensheathed by a basal lamina (MBL), which is specialized at the synaptic site (synaptic BL) and which remains intact following degeneration of the original myofiber. The MBL serves as a scaffold for regenerating myofibers, which form from the fusion of satellite cells that proliferate following damage to the original myofiber. (A) Experiments showing that the synaptic basal lamina contains signals for clustering AChRs and activating AChR genes in synaptic nuclei. Following damage and degeneration of axons, Schwann cells, and myofibers, new myofibers regenerate within the basal lamina of the original myofiber in the absence of the nerve. AChRs cluster and AChR gene expression is reinduced at original synaptic sites in myofibers that regenerate in the absence of the nerve and other original presynaptic cells. (B) Experiments showing that the synaptic basal lamina contains signals for inducing presynaptic differentiation. Following damage and degeneration of axons and myofibers, motor axons regenerate to original synaptic sites in the absence of the myofiber and accumulate synaptic vesicles precisely across from the sites of the original postjunctional folds.

5. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.4 Presynaptic and post-synaptic differentiation are defective in mice lacking MuSK. Whole mounts of muscle from wild-type and MuSK mutant mice were stained with antibodies to neurofilament (NF) and synaptophysin (Syn) to label motor axons and nerve terminals (green), respectively, and with α-bungarotoxin to label acetylcholine receptors (AChRs) (red). In MuSK mutant mice, motor axons fail to stop and differentiate adjacent to the main intramuscular nerve and instead wander aimlessly over the muscle; AChRs, as well as other postsynaptic proteins, are expressed at normal levels but they fail to cluster.

6. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.5 Signaling mechanisms for neuromuscular synapse formation. Agrin, released from motor axon terminals, binds Lrp4, stimulating association between Lrp4 and MuSK and increasing MuSK kinase activity. Phosphorylation of MuSK Y553, in the MuSK juxtamembrane region, causes recruitment and tyrosine phosphorylation of Dok-7, which further stimulates MuSK and recruits Crk and Crk-L adapter proteins to the MuSK/Dok-7 signaling complex. Rac and Rho GTPases function downstream of MuSK/Dok-7/Crk/Crk-L to anchor AChRs at synapses by a poorly understood process that requires Rapsyn, which binds directly to AChRs. The pathway for synapse-specific transcription is less well understood but likely involves JNK-dependent activation of Ets-family transcription factors that stimulate expression of multiple genes encoding synaptic proteins. Synaptic vesicles fuse with the plasma membrane of the nerve terminal, releasing acetylcholine (ACh), which binds to AChRs and causes a conformational change that increases the permeability of the channel to small cations and depolarizes the muscle.

7. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.6 Acetylcholine receptor (AChR) genes are expressed selectively by myofiber synaptic nuclei, leading to enrichment of AChR mRNA in the central, synaptic region of the muscle. A whole mount of a mouse diaphragm muscle was processed for in situ hybridization to reveal the pattern of AChR δ mRNA expression. AChR δ mRNA expression is concentrated in a narrow band in the central, synaptic region of the muscle.

8. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.7 Synapses are tripartite junctions. The electron micrograph shows a parallel fiber synapse in the mouse cerebellum, an example of a glutamatergic synapse. The presynaptic terminal contains a mitochondrion and clusters of synaptic vesicles throughout the terminal. A few vesicles are morphologically docked at the active zone opposite the postsynaptic density. The postsynaptic side is a dendritic spine that in the cross section appears like a circular structure. The electron-dense material opposite the vesicle release sites is the postsynaptic density (PSD). Synaptic cleft and dendritic spine are closely ensheathed by an astroglia process (image courtesy of St é phane Baudouin).

9. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.8 Hypothetical model for the assembly of central synapses. Before synapses formation, axons and dendrites contain synaptic vesicles (SV), active zone components, and neurotransmitter receptors, respectively. Cell – cell contact is initiated via cell surface adhesion and/or signaling molecules (red, t = 0 min). Contact triggers fusion of piccolo-bassoon transport vesicles (PTV, green) with the plasma membrane (t = 15 min), which leads to the deposition of active zone material (black spikes). Subsequently, synaptic vesicles (clear circles) are recruited to the cell – cell contact sites (t = 30 min), and postsynaptic scaffolding molecules (black), NMDA-type (blue), and AMPA type neurotransmitter receptors (red) are delivered sequentially to the maturing postsynaptic membrane. Adapted from Garner et al. (2002).

10. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.9 Synapse-organizing signals at CNS synapses. Different modes of synaptic organizing signals exchanged between pre- and postsynaptic partners and glial cells are illustrated. Wnt7a and FGF22 are retrograde signals derived from the postsynaptic target cell. Transsynaptic adhesion complexes link to scaffolding molecules which interact with presynaptic voltage-gated calcium channels (VGCC) or postsynaptic NMDA-receptors (GluN1/N2), respectively. Several anterograde signals from the presynaptic cell exert their synapse-organizing functions via direct interaction with the amino-terminal-domain (NTD) of glutamate receptors. Perisynaptic astroglia release synapse-organizing molecules such as thrombospondin (TSP). See text for further discussion.

11. Copyright © 2014 Elsevier Inc. All rights reserved. FIGURE 17.10 Model mechanisms for synaptic specificity. (A) In the hippocampus, dentate granule cells (DG) form synapses selectively on pyramidal cells in the CA3 region. Pre- and postsynaptic partners express the same homophilic adhesion molecule (cadherin-9) and thereby preferentially form synapses with each other. (B) In the vertebrate retina, the analysis of laminar specificity of neurite elaboration is facilitated by stereotyped layered organization of the inner plexiform layer (sub-laminae S1-S5). In this system, attractive and repulsive signals have been identified which guide synaptic specificity. Subsets of amacrine and bipolar cells in the inner nuclear layer contain different isoforms of the protein Sidekick (Sdk1 or Sdk2). Through homophilic adhesion, these cells form laminaspecific synaptic connections on the dendrites of Sdk1 or Sdk2-positive ganglion cells, respectively. In the same system, local repulsion is used as an additional specificity mechanism. The neurites elaborated by PlexinA4-positive amacrine cell (PlxA4+) are repelled by semaphorin6A (Sema6A) expressed in sub-laminae S3-S5. Thereby, PlxA4+ neurites are restricted to sub-laminae S1 and S2. Figures adapted from Williams, de Wit, and Ghosh (2010) and Yamagata and Sanes (2008).